By the year 2020 the most common form of glaucoma, primary open angle glaucoma, will affect 59 million people causing bilateral blindness in 5.9 million. Decreased drainage through a strainer-like structure called trabecular meshwork is responsible for the elevated eye pressure and worsening, irreversible damage of the optic nerve. This meshwork regulates flow by an unknown mechanism. It is not known whether drainage decreases in glaucoma because of cell loss or because of accumulating material next to these cells. The declining turnover of meshwork cells in glaucoma is poorly understood because tools do not exist to extensively yet selectively ablate these cells to study division and migration. Evidence has emerged that trabecular meshwork stem cells may be directly anterior to this structure and can be activated after injury. The long-term goal behind this application is o develop new therapeutic strategies to address the progressive outflow failure in glaucoma. The objective of this application is to create a de novo outflow structure. The main hypothesis is that outflow failure is primarily of cellular origin and can be corrected by reverse engineering of this structure using stem cells. The ability to lower intraocular pressure by creating a new regulating barrier rather than surgically removing it (destroying the anterior chamber-blood barrier) or shunting fluid into a non-physiological space, has high clinical relevance. As gene transfer, tissue engineering and stem cell technologies have matured, the proposed work has become feasible only now using the following systematic approach. (1) The substrate of outflow resistance will be identified and removed by ablating either the inner or the outer part of the meshwork with a conditionally cytotoxic lentiviral gene therapy vector. The changing outflow will be analyzed. (2) Trabecular meshwork stem cells will be mobilized as ablation will trigger cell division and migration. These stem cells may be of rare, neural crest origin with a unique differentiation potential into highly specialized tissues (sympathetic and parasympathetic nervous systems, cartilage, melanocytes). (3) A de novo meshwork will be engineered by seeding induced pluripotent donor stem cells. These cells may be able to use nanoscale cues of the ablated meshwork to form a new outflow structure. These studies capitalize on the ability to target meshwork cells for ablation and on recent insights into how micro-environmental cues guide differentiation. The results will provide a systematic understanding of trabecular meshwork progenitor cell activation and of requirements for regulated outflow to occur, thereby enabling new therapeutic targets in glaucoma.